SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens. SIFT-seq: VDJ and scRNA-seq of T cells targeting public neoantigens

We developed a modular, high-throughput discovery platform to simultaneously test whether Mut PIK3Ca is immunogenic and to retrieve paired a/b TCR gene sequences that confer specificity to this NeoAg. This method, termed Stimulation Induced Functional TCR sequencing (SIFT-seq), combines single-cell (sc) TCR V(D)J and transcriptome sequencing. Here, microwell cultures of in vitro stimulated T cells with confirmed neoantigen-specific recognition are selected for SIFT-seq. Matched aliquots of selected wells are acutely stimulated with autologous antigen-presenting cells presenting WT or Mut PI3Ka and the transcriptomic profile of individual clonotypes is assessed to identify neoantigen-specific T cells and retrieve their TCR gene sequences. Overall design: Naïve CD8+ T cells from healthy donor samples were stimulated in vitro with mutated PIK3CA. Following a qPCR screen to identify mutation-specific upregulation of inflammatory transcript, positive wells were selcted for SIFT-seq. Matched aliquots were restimulated acutely with antigen-presenting wells expressing WT or mutated PIK3CA and subject to single-cell RNA-seq and V(D)J immunoprofiling using the 10x genomics platform. All clonotypes associated with a mutation-specific activation signature were identified and their TCR gene sequences were retrieved. Candidate TCR gene sequences were retrovirally transferred to open-repertoire T cell to confirm mutation-specific recognition. Note from submitter: The FASTQ files are not supplied because the data was generated from human patients/donors (patient privacy).